Novel real-time rt-pcr for the sensitive detection of multiple mage gene transcripts

ABSTRACT

The present invention relates to a highly sensitive real-time RT-PCR method for specifically detecting the expression of more than one MAGE gene. The present invention further relates to a diagnostic composition for carrying out such a real-time RT-PCR as well as to oligonucleotides suitable for the cDNA synthesis reaction prior to real-time PCR amplification of more than one marker from the MAGE gene family. To enable the quantitative measurement of MAGE gene expression in a clinical sample an RT-protocol was invented using very sophisticated non-standard conditions to accomplish real-time PCR amplification of cDNA of several MAGE family members in relation to a comparative normalizing reference gene as internal control.

The present invention relates to a highly sensitive real-time RT-PCRmethod for specifically detecting the expression of more than one MAGEgene. The present invention further relates to a diagnostic compositionfor carrying out such a real-time RT-PCR as well as to oligonucleotidessuitable for the cDNA synthesis reaction prior to real-time PCRamplification of more than one marker from the MAGE gene family.

The MAGE gene family was originally described in melanoma patients whencytolytic lymphocytes specific for the MAGE-A1 gene product wereidentified (van der Bruggen, Traversari et al. 1991). This gene waslater found to belong to a cluster of 12 human MAGE-A genes located inthe q28 region of the X chromosome and more recently other members ofthe family characterized as subfamilies MAGE-B, -C and -D were described(Chomez, De Backer et al. 2001). The biological function of MAGE geneproducts is not yet completely understood, but it is assumed that thegenes play an important role in tissue regeneration and differentiation(Old 2001).

Selected members of the MAGE gene family (Table 1) are frequentlyexpressed in many tumors almost irrespective of the histological originbut are completely silent in normal adult tissue with the singleexception of testicular germ cells (De Plaen, Arden et al. 1994).Several MAGE gene products have been identified as promising targets fortumor immunotherapy and have already been used in vaccination trials.The gene products of MAGE-A1, -A2, -A3, -A4, -A6, -A10 and -A12 arefrequently found to induce a cytolytic T-cell response in tumor patientsand are therefore the most promising candidates to serve as specificindicators for cancer.

Recently, the exceptionally restricted expression of MAGE-A genes wasexploited to develop a highly sensitive and tumor-specific multimarkernested RT-PCR based on the independent conventional amplification ofMAGE-A1, -2, -3/6, -4 and -12, respectively (WO 98/46788). This approachwas successfully applied for the sensitive detection of raredisseminated tumor cells in blood and bone marrow of various tumorpatients with many different types of cancer (Kufer 2002). Others haveestablished sensitive conventional MAGE-PCR methods by use of consensusoligonucleotides that coamplify cDNA of several different MAGE genes(Park, Kwon et al. 2002). Such a pan-MAGE-PCR may also detect raredisseminated tumor cells with high sensitivity. However, it does notprovide information on the MAGE gene expression pattern in individualcancer patients, as obtained with a multimarker MAGE PCR specificallyamplifying several different individual members of the MAGE family.

Over the past decade the PCR technology made substantial progressthrough the development of rapid thermocylers and the introduction offluorescence monitoring of amplified products after each cycle, enablingthe quantification of gene expression with “rapid-cycle real-time PCR”assays (e.g. LightCycler® System, ABI PRISM® Sequence Detection System).Sensitive quantification of gene expression thereby relies on thedetection of increasing fluorescence during the exponential phase of PCRproportional to the amount of nucleic acids in the sample at thebeginning of the reaction. Quantification is based on the thresholdcycle (C_(T)-value), the first cycle with detectable fluorescence, andcan be performed in absolute manner with external standards or inrelative manner with a comparative normalizing reference gene serving asinternal calibrator. The determination of a non-inducable reference geneis a critical issue in real-time PCR, since even marginal variations ingene expression will inevitably alter the relative quantificationprofile of the target gene. Usually genes like glyceraldehyd-3-phospatdehydrogenase (GAPDH), porphobilinogen desaminase (PBGD),beta2-microglobin or beta-actin are frequently used as internalcalibrators in real-time PCR. In comparison to conventional endpoint PCRthe real-time assays display even higher sensitivity and precision aswell as a shorter turnaround time for rapid analysis of the results. Ingeneral the fluorescence can be detected sequence-specific by use ofhybridization probes or TaqMan® probes or sequence-unspecific by use ofthe SYBR Green I dye.

Recently, single MAGE markers have been amplified by use of thereal-time PCR technique: Scanlan and coworkers (Scanlan, Gordon et al.2002) investigated MAGE-A3 gene expression in tumor tissue by designinga gene-specific TaqMan probe for measuring mRNA quantity using an ABI7700 sequence detection system. The group of Yoshioka (Yoshioka,Fujiwara et al. 2002) developed a real-time PCR including again theamplification of MAGE-A3 mRNA to screen for tumor cells in resectedlymph nodes of cancer patients. Until today, however, there is noreal-time PCR for utilizing the gene expression of multiple MAGE markersfor quantification of minimal residual tumor disease in cancer patientswho have undergone successful treatment of their primary tumor but areat risk of developing distant metastasis growing from the seed of earlydisseminated tumor cells; these patients, whose tumor load can beadvantageously determined by the method of the present invention,urgently need an adjuvant tumor therapy to prevent the seed ofmetastasis from growing.

The investigation of only single markers is bound to result in adramatic loss of sensitivity, because of the expression heterogeneity ofmalignant tumors in general and of single disseminated tumor cells inparticular. For these reasons a multimarker real-time RT-PCR method forthe highly sensitive detection of multiple tumor relevant markersselected from the MAGE family, like e.g. MAGE-A1, -A2, -A3, -A4, -A6,-A10 and -A12, was highly preferable.

Accordingly, the present invention relates to a highly sensitivereal-time RT-PCR for the specific and reliable detection of mRNAtranscribed by rare tumor cells from more than one MAGE gene. On the onehand, PCR-primers described in the prior art (Kufer 2002; Park, Kwon etal. 2002) for conventional highly sensitive pan- or multimarker-MAGEamplification methods are also applicable for highly sensitive real-timeRT-PCRs. On the other hand, however, methods for reverse transcriptionof mRNA as successfully used for conventional highly sensitive pan- ormultimarker-MAGE RT-PCRs—unexpected from the prior art—turned out to beinsufficient for corresponding highly sensitive real-time RT-PCRs.

Since those RT-PCRs designed to detect rare tumor cells, which rely onthe amplification of a single marker gene only, are particularlysusceptible to expression loss or down-regulation connected with thegenomic instability and phenotypic heterogeneity of tumor cells,reliable detection of at least two different MAGE gene transcripts bythe real-time RT-PCR of the present invention was absolutely required.Therefore, it is essential to make sure that each single member of theMAGE family selected as a marker for the highly sensitive real-timeRT-PCR is reliably converted from mRNA to cDNA by reverse transcriptionwith reproducible efficiency. Only under this prerequisite the relativecontent in biological samples to be analyzed of the different MAGE mRNAspecies compared to each other can be determined or quantification ofthe different MAGE transcripts carried out in comparison to an internalcalibrator template like the PBGD mRNA.

As a solution to this technical problem it has been found in the presentinvention, that reverse transcription of the different MAGE transcriptsand optionally of the calibrator mRNA must be carried out simultaneouslyin a single cDNA-synthesis reaction, using highly selectedoligonucleotide primers and sophisticated reaction conditions forreverse transcription, which could not be anticipated from the priorart.

Accordingly the present invention relates to a highly sensitivereal-time RT-PCR capable of specifically detecting the expression ofmore than one MAGE gene, wherein reverse transcription of thecorresponding MAGE transcripts is carried out simultaneously in a singlecDNA-synthesis reaction. Carrying out efficient and reliable reversetranscription of different transcripts in a single cDNA-sythesisreaction was no trivial task, because the methods for reversetranscription (RT) of two or more different MAGE transcripts, whichaccording to the prior art led to the reliable conversion of each singlespecies of MAGE-mRNA into sufficient cDNA detectable by highly sensitiveconventional PCRs, suprisingly failed to do so in combination with ahighly sensitive real-time MAGE-PCR. Neither unspecific reversetranscription using cDNA-priming with oligo-dT or random hexanucleotidesnor specific cDNA-priming with an established combination of mono- anddual-specific oligonucleotides hybridizing to the differentMAGE-transcripts, respectively, proved to be sufficient for obtainingspecific amplification products at the desired high sensitivity levelfor each member of the MAGE family selected as marker for the subsequentreal-time PCR (Example 2). Even the use of a “pan-MAGE-primer” forcDNA-synthesis as taught by the closest prior art. (WO 98/46788)disclosing a highly sensitive conventional multimarker MAGE RT-PCR didnot succeed under standard conditions in sufficient reversetranscription of mRNA from every single MAGE gene used as marker in thecorresponding real-time multimarker MAGE RT-PCR. Thus, it is required,in accordance with the present invention, to identify by careful testingunder many different reaction conditions one or more primers for reversetranscription (=RT-primer), each hybridizing to the mRNA of one or moredifferent members of the MAGE gene family. It is essential that thetesting of RT-primers for the highly sensitive real-time MAGE RT-PCR iscarried out in the presence of the whole cocktail of RT-primers duringcDNA-synthesis. This is required because of frequent interferences amongdifferent RT-primers, which are neither predictable from cDNA-synthesisreactions with only one RT-primer nor from the teachings of the priorart.

A real-time MAGE PCR, i.e. PCR-amplification of reverse transcribedMAGE-cDNA, in accordance with the present invention, can be implementedeither by using a sequence unspecific DNA-dye like SYBR Green I or byapplying sequence-defined fluorescent probes for the detection ofspecific amplicons. For carrying out the latter method, the sequences ofMAGE mRNA molecules have to be screened for unique marker-definingregions if the detection of individual MAGE parameters is desired orpan-MAGE specific areas if the detection of several MAGE markers isdesired in a single reaction. This unique hybridization region on thesequence must be located in between two oligonucleotides used as primersfor the PCR and should neither be self-complementary, monotonous, orrepetitive nor complementary to the PCR primers. The application ofTaqMan probes requires the design of a single double-labeled fluorescentprobe, for the application of hybridization probes the design of twofluorescent oligonucleotides is needed that hybridize in close proximity(1 to 5 bases) to each other on the amplicon to enable thedistance-dependent transfer of energy between the fluorophores(fluorescence resonance energy transfer). The reaction conditions haveto be carefully evaluated and optimized, involving the adaptation ofprimer and probe concentrations, temperatures and duration ofPCR-cycling etc.

In a preferred embodiment of the method of the invention the MAGE genesserving as markers in a highly sensitive real-time RT-PCR are selectedfrom the functional genes of MAGE subfamilies A, B and/or C (Table 1).Except for the pseudogenes, expression of the members of theseMAGE-subfamilies is highly restricted to tumor cells, while they arecompletely silent in normal adult tissue with the only exception oftesticular germ cells. Thus expression of functional MAGE A, B and/or Cgenes as detected by the highly sensitive real-time RT-PCR of theinvention in blood, bone marrow, lymph nodes or other secondary organsof a tumor patient is highly indicative of the systemic spread of cancercells from the primary tumor. Because of its quantitative nature, thehighly sensitive real-time RT-PCR of the invention is particularlyuseful for measuring the load of disseminated tumor cells in individualpatients, thus estimating the risk of a metastatic relapse originatingfrom the early tumor cell spread that took place prior to successfultreatment of the primary tumor. Thus, the method of the invention mayhelp to decide more precisely on the requirement of an adjuvant tumortherapy than is possible with diagnostic methods of the prior art.

Besides blood and bone marrow many kinds of body fluids or tissues, likeurine, stool or sputum, are easily accessible to search for malignantcells. The real-time MAGE RT-PCR of the present invention is thereforealso applicable as highly sensitive screening tool in secondary tumorprevention and can achieve the early detection of neoplasia particularlyin individuals who are highly at risk of developing cancer.

In a particularly preferred embodiment of the method of the presentinvention the MAGE genes serving as markers in a highly sensitivereal-time RT-PCR comprise MAGE-A 1, 2, 3, 4, 6, 10 and/or 12. Thesegenes are most frequently expressed in many different types of tumors ofvarious histological origins. Moreover, all members of this selectedgroup of MAGE genes encode target antigens for cytotoxic T cells. Thus,the highly sensitive real-time RT-PCR of the invention willadvantageously provide quantitative MAGE gene expression profiles ofindividual cancer patients as a basis for the rational design ofMAGE-based tumor vaccines. So far, only qualitative patterns of MAGEgene expression could be obtained by using prior art methods making thechoice of MAGE gene products to be included in a tumor vaccine moredifficult.

In another preferred embodiment of the method of the invention at leastone primer for reverse transcription of MAGE mRNA is selected from thefollowing groups of oligonucleotides: primer sequence (5′ - 3′) (A)MgRT1a CCA GCA TTT CTG CCT TTG TGA MgRT1b CCA GCA TTT CTG CCT GTT TGMgRT2 CAG CTC CTC CCA GAT TT MgRT3a ACC TGC CGG TAC TCC AGG MgRT3b ACCTGC CGG TAC TCC AGG TA MgRT4 GCC CTT GGA CCC CAC AGG AA MgRT5a AGG ACTTTC ACA TAG CTG GTT TCA MgRT5b GGA CTT TCA CAT AGC TGG TTT C MgRT6 TTTATT CAG ATT TAA TTT C (B) Mg1_RT1 CAA GAG ACA TGA TGA CTC TC Mg1_RT2 TTCCTC AGG CTT GCA GTG CA Mg1_RT3 GAG AGG AGG AGG AGG TGG C Mg1_RT4 GAT CTGTTG ACC CAG CAG TG Mg1_RT5a CAC TGG GTT GCC TCT GTC Mg1_RT5c CTG GGT TGCCTC TGT CGA G Mg1_RT5d GGG TTG CCT CTG TCG AGT G Mg1_RT5e GGC TGC TGGAAC CCT CAC Mg1_RT6 GCT TGG CCC CTC CTC TTC AC Mg1_RT7 GAA CAA GGA CTCCAG GAT AC

Primers for reverse transcription as depicted in group A, are perfectlymatching with each of the mRNA-sequences of MAGE-A 1, 2, 3, 4, 6, 10 and12. However, most surprisingly, despite the perfect match, none of theseRT-primers alone leads to sensitive detection of MAGE-A 1 expression byreal-time RT-PCR under standard conditions as provided e.g. by themanufacturer of the LightCycler System. Under these recommendedconditions the weakness in detection of MAGE-A 1 expression can only becompensated, as surprisingly found in the present invention, bycombining two primers of group A with each other (e.g. MgRT3a+MgRT5a) ora group A-primer with one of the group B-primers, which are monospecificfor the cDNA-synthesis of MAGE-A 1 only (Example 2). Depending on whichat least two different members of the MAGE-A group encoding targetantigens for cytotoxic T cells (i.e. MAGE-A 1, 2, 3, 4, 6, 10 and 12)are to be detected by real-time RT-PCR, different single RT-primers orcombinations of RT-primers of group A and/or B may be applicable. In anyindividual case, however, in accordance with the present invention,careful testing of candidate RT-primers is required to end up with anoptimal choice allowing the expression of the selected MAGE genes to bedetected by real-time RT-PCR with a high level of sensitivity. Aspointed out above, testing of RT-primers for the highly sensitivereal-time MAGE RT-PCR has to be carried out in the presence of the wholecocktail of RT-primers during cDNA-synthesis, in order to cope with theunpredictable interferences among different RT-primers.

According to the teaching of the prior art (WO 98/46788) the averageexpert may—without undue burden—identify specific primers for thecDNA-synthesis that efficiently hybridize to the mRNA of MAGE-1, -2,-3/6, -4 and -12. However, the whole series of “pan-MAGE primers” asdepicted in group A failed to result in cDNA-synthesis, which would haveallowed the highly sensitive detection of each single marker mRNA byreal-time PCR as carried out according to the recommended protocolprovided by the manufacturer of the LightCycler System (Roche). In everytested case the highly sensitive detection by real-time PCR of at leastone mRNA-species from the group consisting of MAGE-1, -2, -3/6, -4 and-12 failed despite perfect hybridization of every pan-MAGE cDNA-primerto each of the corresponding transcripts (example 2). This clearlydiffers from the teaching of the above mentioned prior art documentreferring to a conventional multimarker MAGE RT-PCR.

In a further particularly preferred embodiment of the method of thepresent invention, in addition to the reverse transcription of MAGEtranscripts, reverse transcription of a calibrator mRNA issimultaneously carried out in the same single cDNA-synthesis reactionfollowed by PCR-ampliflcation of MAGE- and calibrator cDNAs. For makingthe quantitative results obtained by analysis of different blood-, bonemarrow- or other tissue samples from one or more cancer patients usingthe real-time MAGE RT-PCR comparable with each other, a normalizingreference gene, in accordance with the present invention, is preferablyincluded in the assay. In order to be capable of serving as internalcalibrator the normalizing reference marker most preferably is anessentially non-inducible gene. It is further preferred that theexpression level of the reference gene is comparable to the targetgene(s) and constant in essentially all cells of the sample.Furthermore, in accordance with the present invention, it is criticalthat a specific cDNA-primer for reverse transcription (RT) of thecalibrator mRNA is used as integral member of the RT-primer cocktailcomprising the MAGE-specific cDNA-primers to guarantee equal assayconditions for both the different MAGE transcripts to be analysed andthe reference marker.

In another preferred embodiment of the method of the present inventionthe normalizing reference gene serving as internal calibrator isporphobilinogen desaminase (PBGD), glyceraldehyd-3-phospat dehydrogenase(GAPDH), beta-2-microglobin or beta-actin.

In a further preferred embodiment of the method of the present inventionthe primer for reverse transcription of PBGD mRNA is selected from thefollowing group of oligonucleotides: primer sequence (5′ - 3′) PBGD_RT2CAT ACA TCG ATT CCT CAG GGT PBGD_RT3 GAA CTT TCT CTG CAG CTG GGCPBGD_RT4 TGG CAG GGT TTC TAG GGT CT PBGD_RT10a GGT TTC CCC GAA TAC TCCTG PBGD_RT10d TTG CTA GGA TGA TGG CAC TG PBGD_RT12b CCA AGA TGT CCT GGTCCT TG PBGD_RT12c CAG CAC ACC CAC CAG ATC PBGD_RT12d AGA GTC TCG GGA TCGTGC PBGD_RT12e AGT CTC GGG ATC GTG CAG PBGD_RT12f TCT CGG GAT CGT GCAGCA PBGD_RT12g ATG CAG CGA AGC AGA GTC T PBGD_RT12h CCT TTC AGC GAT GCAGCG PBGD_RT13a GTA TGC ACG GCT ACT GGC PBGD_RT14a GCT ATC TGA GCC GTCTAG AC PBGD_RT15a AAT GTT ACG AGC AGT GAT GC PBGD_RT15b TGG GGC CCT CGTGGA ATG PBGD_RT15e CAG TTA ATG GGC ATC GTT AAG PBGD_RT15f ATC TGT GCCCCA CAA ACC AG PBGD_RT15g GGC CCG GGA TGT AGG CAC PBGD_RT15h GGT AAT CACTCC CCA GAT AG PBGD_RT15i CTC CCG GGG TAA TCA CTC PBGD_RT15j CAG TCT CCCGGG GTA ATC PBGD_RT15k TGA GGA GGC AAG GCA GTC PBGD_RT15l GGA TTG GTTACA TTC AAA GGC

For each individual real-time MAGE RT-PCR, however, in accordance withthe present invention, careful testing of candidate RT-primers specificfor PBGD or the mRNA of another reference gene together with thecandidate MAGE RT-primer(s) is required to end up with an optimal choiceallowing the expression of the selected MAGE genes to be measured byreal-time RT-PCR in comparison with reliable expression signals from thereference gene at a high level of sensitivity. Also in this case,testing of RT-primers for the highly sensitive real-time MAGE RT-PCRincluding the RT-primer for reverse transcription of the internalcalibrator mRNA has to be carried out in the presence of the wholecocktail of RT-primers during cDNA-synthesis, in order to cope with theunpredictable interferences among different RT-primers.

In another embodiment of the method of the present invention thePCR-primers for amplification of PBGD-cDNA comprise oligonucleotidesselected from the following groups: PBGD sequence (5′ - 3′) sense primerhu_PBGD_se AGA GTG ATT CGC GTG GGT ACC PBGD_8 GGC TGC AAC GGC GGA AGAAAA C PBGD_8_F TGC AAC GGC GGA AGA AAA C PBGD_ATG-Eco ATG TCT GGT AACGGC AAT GC antisense primer PBGD_3 TTG CAG ATG GCT CCG ATG GTG AAPBGD_3.1_R GGC TCC GAT GGT GAA GCC PBGD_R TTG GGT GAA AGA CAA CAG CAT C

In an even more preferred embodiment of the method of the presentinvention oligonucleotides hu_PBGD_se and PBGD_(—)3.1_R or hu_PBGD_seand PBGD_R are used as primer pairs for PCR-amplification of PBGD-cDNA.

Actually, to introduce PBGD as internal calibrator for quantification ofMAGE transcripts by a highly sensitive real-time RT-PCR according to thepresent invention 24 different PBGD-specific cDNA-primers were designedand tested in the presence of the MAGE-specific cDNA-primers forefficient reverse transcription of PBGD-mRNA in a PBGD-specificreal-time RT-PCR. Those PBGD-specific cDNA-primers, which gave goodresults in the subsequent PBGD-amplification by real-time PCR were thentested in combination with the MAGE-specific cDNA-primers in aquantitative multimarker MAGE real-time RT-PCR. However, it was foundthat no combination of three cDNA-primers each consisting of a pan-MAGE-and a MAGE-A1 specific cDNA-primer plus a PBGD-specific cDNA-primer ledto the highly sensitive amplification of every single marker from thegroup consisting of MAGE-A1, -2, -3/6, -4, -10 and -12 transcripts byreal-time RT-PCR (example 3). In order to solve this problem it turnedout, that the reduction of cDNA-primers from a triple to a doublecombination was inevitable. For this purpose we had to invent anRT-protocol using very sophisticated non-standard conditions comprisingunusual primer concentrations and the use of a highly selectedpolymerase to make a single pan-MAGE cDNA-primer work together with aPBGD cDNA-primer, without loosing the highly sensitive amplification ofonly a single marker in the subsequent real-time PCR (example 3). Thiseventually led to a final protocol for a highly sensitive quantitativemultimarker MAGE real-time RT-PCR which by no means could have beenanticipated from the prior art (example 5).

Accordingly, a highly preferred embodiment of the method of the presentinvention is related to the use of not more than two differentoligonucleotides (including the RT-primer of an internal calibrator) asprimers for reverse transcription in the cDNA-synthesis reaction of thereal-time MAGE RT-PCR of the present invention.

In a most preferred embodiment of the method of the present inventionoligonucleotides MgRT3a and/or Mg1_RT5a are used as primers for reversetranscription in the cDNA-synthesis reaction.

In a further most preferred embodiments of the method of the presentinvention oligonucleotides MgRT3a and PBGD_RT15b are used as primers forreverse transcription in the cDNA-synthesis reaction.

In another embodiment of the method of the present invention the MAGE-and/or the calibrator-PCR are nested or semi-nested PCRs. In order toachieve the desired high sensitivity for detection of mRNA transcribedby rare tumor cells from more than one MAGE gene, the real-time RT-PCRmay be designed as nested or semi-nested PCR. For this purpose a firstround of cDNA-amplification may be carried out with an appropriate pairof PCR-primers either by conventional or real-time PCR. Most preferably,this first round of PCR should not proceed to the plateau phase ofamplification. Otherwise, quantification of the template content in thesample to be analyzed by the method of the present invention may becomevery difficult or even impossible. Moreover, it may be preferable tostop such a first round of PCR in the early or middle linear phase ofamplification instead of proceeding to the late linear phase, in orderto avoid interferences of an excess of preamplified PCR-products withthe subsequent round of real-time PCR. Accordingly, the number ofPCR-cycles and the reaction conditions that are appropriate for such apreamplification step have to be carefully optimized, respectively. Inparticular these parameters should be adapted to the distribution oftemplate amounts in the collection of samples to be analyzed, to makesure, on the one hand, that the level of high sensitivity of the methodof the invention is sufficient to detect MAGE in those samples showingvery weak expression and, on the other hand, that quantification of MAGEin other samples showing higher expression is still feasible.

In a particularly preferred embodiment of the method of the presentinvention PCR-primers are used comprising pairs of oligonucleotidesspecifically amplifying only a single member of the selected group ofMAGE genes, respectively. Despite the high homology among differentmembers of the MAGE gene family, making the design of such monospecificoligonucleotides more difficult, a highly sensitive real-time MAGERT-PCR for detecting the individual expression of more than one MAGEgene is highly preferable. Only thus, a quantitative expression profileof individual MAGE genes of rare disseminated tumor cells in individualcancer patients can be obtained, which may be essential for theselection of those members of the MAGE family to be included in anoptimal tumor vaccine. Moreover, the prognostic impact of the expressionlevels of single members of the MAGE gene family may vary with differenttypes of cancer, which can be analyzed with the real-time RT-PCR of thepresent invention only when pairs of PCR-primers monospecific for thecDNA of individual MAGE genes are used that do not crossamplify othermembers of the MAGE family.

In another embodiment of the method of the present invention PCR-primersare used comprising pairs of oligonucleotides amplifying more than onemember of the selected group of MAGE genes, respectively (=pan-MAGEPCR).

Following reverse transcription real-time PCR amplification of MAGE cDNAwith such consensus primers, like those suggested by Park et al. (Park,Kwon et al. 2002) may be carried out, which make use of the high levelof sequence homology among the different MAGE gene transcripts. Usingthis or similar approaches real-time RT-PCR may lead toMAGE-amplification products derived from the transcripts of MAGE-A 1,-2, -3, -4, -6 and/or 12 expressed by as few as five cancer cells (e.g.from the human colon cancer cell line HT-29) in 10 ml of blood, whilestaying negative with blood from healthy donors. For detection of thereal-time PCR-amplification product(s) the sequence-independent SYBRgreen I method can be applied using the LightCycler System;alternatively, sequence-specific fluorescent probes, e.g. TaqMan orhybridization probes may be used. Furthermore, tissue samples (e.g.bronchoscopic biopsies) from cancer patients with different types oftumors (e.g. non-small cell lung (NSCL) cancer) may be analyzedaccordingly. For this embodiment of the method of the invention it is ofparticular advantage that the particular way of cDNA-synthesis disclosedby the present invention makes sure that each single member of the MAGEfamily selected as a marker for the highly sensitive real-time RT-PCR isreliably converted from mRNA to cDNA by reverse transcription withreproducible efficiency, because due to coamplification of cDNA fromdifferent MAGE genes drop-outs of single markers at the stage of reversetranscription may easily remain unrecognized in the PCR e.g. by apositive signal derived from only one marker thus pretending successfuldetection of other presumably coamplified markers that indeed may havefailed sufficient cDNA-synthesis although being expressed.

In another particularly preferred embodiment of the method of thepresent invention the PCR-primers for amplification of MAGE-cDNAcomprise oligonucleotides selected from one of the following groups:PCR-primer sequence (5′ - 3′) (C) MAGE-A1 GTA GAG TTC GGC CGA AGG AACMAGE-A1 CAG GAG CTG GGC AAT GAA GAC MAGE-A2 CAT TGA AGG AGA AGA TCT GCCT MAGE-A2 GAG TAG AAG AGG AAG AAG CGG T MAGE-A3/6 GAA GCC GGC CCA GGCTCG MAGE-A3/6 GAT GAC TCT GGT CAG GGC AA MAGE-A4 CAC CAA GGA GAA GAT CTGCCT MAGE-A4 TCC TCA GTA GTA GGA GCC TGT MAGE-A10 CTA CAG ACA CAG TGG GTCGC MAGE-A10 GCT TGG TAT TAG AGG ATA GCA G MAGE-A12 TCC GTG AGG AGG CAAGGT TC MAGE-A12 ATC GGA TTG ACT CCA GAG AGT A (D) MAGE-A1 TAG AGT TCGGCC GAA GGA AC MAGE-A1 CTG GGC AAT GAA GAC CCA CA MAGE-A2 CAT TGA AGGAGA AGA TCT GCC T MAGE-A2 CAG GCT TGC AGT GCT GAC TC MAGE-A3/6 GGC TCGGTG AGG AGG CAA G MAGE-A3/6 GAT GAC TCT GGT CAG GGC AA MAGE-A4 CAC CAAGGA GAA GAT CTG CCT MAGE-A4 CAG GCT TGC AGT GCT GAC TCT MAGE-A10 ATC TGACAA GAG TCC AGG TTC MAGE-A10 CGC TGA CGC TTT GGA GCT C MAGE-A12 TCC GTGAGG AGG CAA GGT TC MAGE-A12 GAG CCT GCG CAC CCA CCA A

This embodiment of the invention is advantageous because it is capableof measuring the individual expression of all those members of theMAGE-A subfamily encoding target antigens recognized by cytotoxic Tlymphocytes, which are thus relevant for tumor vaccination. In theparticular case of MAGE-A3 and 6, which are amplified by the same pairsof PCR-primers depicted in group C and D, there is no loss ofinformation relevant for vaccine design caused by the coamplification,because the proteins encoded by MAGE-A3 and 6 are almost identical dueto a sequence homology of 99%.

In an even more preferred embodiment of the method of the presentinvention primers of group C are used for a first round and/or primersof group D for a second round of PCR-amplification.

This embodiment of the invention is advantagous for carrying out ahighly sensitive nested or semi-nested real-time MAGE RT-PCR.

In another embodiment of the method of the present invention a single ordouble pair of PCR-primers is used amplifying all members of theselected group of MAGE genes, respectively. This embodiment relates to ahighly sensitive real-time RT-PCR specifically detecting the expressionof more than one MAGE gene, by a single pair of pan-MAGE PCR-primers incase of a single-step PCR or a double pair of pan-MAGE PCR-primers incase of a nested or semi-nested PCR. Due to the high level of sequencehomology among the different MAGE genes, sites of sequence identitybetween all members of a selected group of MAGE genes may be found bycomputer-based sequence analysis, where such pan-MAGE PCR-primers canhybridize.

As with every pair of PCR-primers, either monospecific for the cDNA ofan individual MAGE gene or oligospecific for the cDNAs of some or allmembers of a certain group of MAGE genes (=pan-MAGE PCR-primer), primerpositions have to be selected in a way to avoid amplification of genomicMAGE DNA. For example, amplification of genomic MAGE-sequences can beavoided by the use of primers localized in different exons or primersspanning different neighboring exons, thus restricting hybridization tocDNA only. Furthermore, the positions of the PCR-primers have to bechosen to fall within the sequence segment(s) of the MAGE transcript(s),which is (are) reverse transcribed by the actual RT-primer(s) used forcDNA-synthesis.

The present invention further relates to a diagnostic compositioncomprising one or more suitable cDNA-primers for simultaneous reversetranscription of more than one different MAGE gene transcripts andoptionally an appropriate calibrator mRNA in a single cDNA-synthesisreaction. The diagnostic composition of the invention is particularlyuseful for carrying out a variety of highly sensitive real-time MAGERT-PCRs, thus allowing the quantification of the tumor cell load incancer patients suffering from systemic tumor cell spread, by measuringthe content of more than one kind of MAGE mRNA in blood-, bone marrow-,lymph node or other tissue samples. Moreover, the diagnostic kit isparticularly useful for determining quantitative MAGE gene expressionprofiles of rare disseminated tumor cells in individual cancer patients,thus allowing the rational design of a MAGE-based tumor vaccine. Inaccordance with the present invention it is particularly preferable thatat least one cDNA-primer of the diagnostic composition is MgRT3a,Mg1_RT5a or PBGD_RT15b.

Finally the present invention also relates to an oligonucleotideselected from the following group of primers:

MgRT3a

Mg1_RT5a

PBGD_RT15b

In accordance with the present invention, it was found that theseoligonucleotides are particularly useful for simultaneously priming thereverse transcription of mRNA from more than MAGE genes in a singlecDNA-synthesis reaction. It has been further found, in accordance withthe present invention, (1) that this “single-pot” cDNA-synthesis isessential to make sure that each single member of the MAGE familyselected as a marker for the highly sensitive real-time RT-PCR of theinvention is reliably converted from mRNA to cDNA by reversetranscription with reproducible efficiency and (2) that only under thisprerequisite the relative content in biological samples to be analyzedof the different MAGE mRNA species compared to each other can bedetermined or quantification of the different MAGE transcripts carriedout in comparison to an internal calibrator template like the PBGD mRNA.

Definitions

The term

-   “RT” or “cDNA synthesis” is used in the current invention for the    conversion of mRNA into complementary DNA (cDNA) by a reverse    transcriptase enzyme in a reverse transcription reaction (RT).-   “RT-PCR” is used in the current invention for methods applying a    polymerase chain reaction (PCR) after converison of mRNA into    complementary DNA (cDNA) by a reverse transcription reaction (RT).-   “conventional PCR” is used in the current invention for    non-fluorescent PCR methods operated on all kinds of traditional    thermocyclers.-   “nested PCR” is used in the current invention for PCR methods    comprising two amplification steps with different sets of primers    for the first and second round of amplification.-   “semi-nested PCR” is used in the current invention for PCR methods    comprising two amplification steps with one shared primer for the    first and second round of amplification.-   “real-time PCR” is used in the current invention for    fluorescence-based PCR methods on photometric thermocyclers with the    option for quantification of original template amounts. The method    can include additional preamplification steps on a traditional    thermocycler for a defined number of PCR-cycles.-   “multimarker MAGE PCR” is used in the current invention for PCR    assays that enable the separate amplification of cDNA of different    individual MAGE genes.-   “pan MAGE PCR” is used in the current invention for PCR assays that    enable the amplification of cDNA of different MAGE genes by one or    more pairs of consenus PCR-primers each capable of coamplifying at    least two different MAGE gene transcripts.-   “RT-primer” or “cDNA synthesis primer” is used in the current    invention for oligonucleotides designed to hybridize only to a    defined target mRNA to yield specific cDNA molecules of these    transcripts in a reverse transcription reaction.-   “PCR primer” is used in the current invention for oligonucleotides    designed to hybridize only to certain regions of target cDNA to    yield amplicons of a specific length in a PCR reaction.-   “high sensitivity” is used in the current invention for the    capability of a PCR method to yield detectable MAGE specific    amplificates from 5 or less tumor cells in 2 ml of whole blood.    Additionally a crossing point below 30 PCR-cycles is required for    real-time PCR-methods to fulfil the definition.

REFERENCES

-   Chomczynski, P. and N. Sacchi (1987). “Single-step method of RNA    isolation by acid guanidinium thiocyanate-phenol-chloroform    extraction.” Anal Biochem 162(1): 156-9.-   Chomez, P., O. De Backer, et al. (2001). “An overview of the MAGE    gene family with the identification of all human members of the    family.” Cancer Res 61(14): 5544-51.-   De Plaen, E., K. Arden, et al. (1994). “Structure, chromosomal    localization, and expression of 12 genes of the MAGE family.”    Immunogenetics 40(5): 360-9.-   Kufer, P., Zippelius, A., Lutterbuese, R., Mecklenburg, I., Enzmann,    T., Montag, A., Weckermann, D., Passlick, B., Prang, N., Reichardt,    P., Dugas, M., Kollermann, M. W., Pantel, K., Riethmuller, G.    (2002). “Heterogeneous Expression of MAGE-A Genes in Occult    Disseminated Tumor Cells: a novel multimarker RT-PCR for diagnosis    of micrometastatic disease.” Cancer Res 62: 251-261.-   Old, L. J. (2001). “Cancer/Testis (CT) antigens—a new link between    gametogenesis and cancer.” Cancer Immunity 1((30 Mar. 2001)): 1.-   Park, J. W., T. K. Kwon, et al. (2002). “A new strategy for the    diagnosis of MAGE-expressing cancers.” J Immunol Methods 266(1-2):    79-86.-   Scanlan, M. J., C. M. Gordon, et al. (2002). “Identification of    cancer/testis genes by database mining and mRNA expression    analysis.” Int J Cancer 98(4): 485-92.-   Serrano, A., B. Lethe, et al. (1999). “Quantitative evaluation of    the expression of MAGE genes in tumors by limiting dilution of cDNA    libraries.” Int J Cancer 83(5): 664-9.-   van der Bruggen, P., C. Traversari, et al. (1991). “A gene encoding    an antigen recognized by cytolytic T lymphocytes on a human    melanoma.” Science 254(5038): 1643-7.-   Yoshioka, S., Y. Fujiwara, et al. (2002). “Real-time rapid reverse    transcriptase-polymerase chain reaction for intraoperative diagnosis    of lymph node micrometastasis: clinical application for cervical    lymph node dissection in esophageal cancers.” Surgery 132(1): 34-40.

For Information on Standard Conditions Known in the Prior Art theFollowing References May be Used

-   Sambrook, J., Russel, D., W. (2001). “Molecular Cloning—A Laboratory    Manual.” 3^(rd) edition 2001, Cold Spring Harbor Laboratory Press,-   Roche Molecular Biochemicals (2000). “LightCycler Operator's    Manual.” Version 3.5-   Meuer, S., Wittwer, C., Nakagawara, K., I. (Eds.) (2001). “Rapid    Cycle Real-Time PCR—Methods and Applications.” Springer Publishing-   Dietmaier, W., Wiftwer, C., Sivasubramanian, N. (Eds.) (2002).    “Rapid Cycle Real-Time PCR—Methods and Applications—Genetics and    Oncology.” Springer Publishing-   http://wvw.roche-applied-science.com/lightcycler-online-   http://www.appliedbiosystems.com/techsupport

FIGURE LEGENDS

FIG. 1: Real-time amplification plot (A) and melting curve analysis (B)of MAGE-A1 transcripts in 2 ml of blood spiked with different numbers ofMz2-Mel cells as indicated using a standard LightCycler-DNA Master SYBRGreen I protocol after oligo-dT primed cDNA synthesis. The arrows in Bindicate the maximum of product dissemination over the indicatedtemperature range. The specific MAGE-A1 PCR product displays a meltingpeak at approximately 88.8° C. Unspecific products, e.g. primer dimersin this case, can be identified by their different dissociation curve.The gel electrophoresis (C) confirms specific amplification of thetranscript and reliable detection of 1 tumor cell in 2 ml of wholeblood.

FIG. 2: Melting curve analysis after completion of a standardLightCycler-DNA Master SYBR Green I protocol for MAGE-A2 (A) andMAGE-A12 (B) after cDNA synthesis with oligo-dT priming. At least 10tumor cells in 2 ml of blood are required for the generation of specificPCR products, samples with lower cell numbers do not result in specificsignals with this protocol.

FIG. 3: Real-time amplification plot (A) and melting curve analysis (B)of MAGE-A4 mRNA in 2 ml of blood spiked with different numbers of LB-SARcells as indicated. The first round of the nested PCR was performed with15 cycles. Samples spiked with 5 and 10 LB-SAR cells yield the specificMAGE-A4 PCR product displaying a melting peak of approximately 87.6° C.Unspecific products obtained when spiking 1 cell or no cells can bedistinguished by their different melting curves.

FIG. 4: Real-time amplification plot (A) and melting curve analysis (B)of the same RNA sample applied in FIG. 3 after 20 cycles of first PCR.The extension of preamplification leads to an improved sensitivity levelwith specific detection of MAGE-A4 PCR products when 1 LB-SAR tumor cellwas diluted in 2 ml of blood. This positive achievement was associatedwith the increased formation of primer dimers in negative controls.

FIG. 5:—Real-time amplification plot of MAGE transcripts after cDNAsynthesis with a combination of antisense PCR primers for MAGE-1, -2,-3/6, -4 and -12. The application of 5 specific oligonucleotides in thereverse transcription reaction led to the formation of severalunspecific products in the consecutive real-time PCR, e.g. primerdimers, associated with a deformation of the amplification curve.

FIG. 6: Melting curve analysis of MAGE-A10 PCR products obtained withdifferent primer-combinations for preamplification. Specific MAGE-A10products are detectable only when a selection of two sense primers isused with a single antisense primer (Mg10_anse5). Approaches with theantisense primer Mg10_anse4 for preamplification do not result inspecific signals, although this oligonucleotide could be successfullyapplied as antisense primer in real-time PCR.

The examples illustrate the invention.

EXAMPLE 1 Limitation of Oligo-dT Primed cDNA Synthesis for DetectingMAGE Transcripts Expressed by Rare Tumor Cells in Blood with a Real-TimeMultimarker MAGE-PCR

The first evaluation of a real-time MAGE PCR was performed in tumor celldilution experiments. For this purpose we spiked 2 ml whole blood ofhealthy donors with different numbers of Mz2-Mel cells for amplificationof MAGE-1, -2, -3/6 and -12 transcripts. To avoid degradation of the RNAeach sample was immediately mixed with 10 ml denaturating nucleic acidextraction buffer [4 M guanidine isothiocyanate, 0.5% sarcosyl(N-laurylsarcosine sodium salt), 25 mM sodium citrate (pH=7.0), 0.7%2-mercaptoethanol]. Total RNA was isolated according to the method ofChomczynski and Sacchi (Chomczynski and Sacchi 1987) and wasmeasured-spectrophotometrically cDNA was synthesized from 1 μg of totalRNA by extension with 1.6 μg oligo-dT primer and 20 U avianmyeloblastosis virus reverse transcriptase (Roche MolecularBiochemicals, Mannheim, Germany) at 25° C. for 10 min., 42° C. for 60min. and 99° C. for 5 min.

A first PCR round for preamplification was performed in 50 μl reactionscontaining 5 μl of cDNA, 5 μl of 10×PCR buffer (200 mM Tris, pH=8.0, 500mM KCl), 1.5 μl of MgCl₂ (50 μM), 4 μl of each dNTP (100 μM)(Invitrogen, Groningen, Netherlands), 0.2 μM of each of the outer MAGEprimers, and 1.25 units of Platinum Taq DNA-Polymerase (Invitrogen) andwas run on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City,USA) according to the following cycle profile: enzyme activation at 95°C. for 3 min; denaturation at 95° C. for 30 s, annealing at 60° C. for45 s and extension at 72° C. for 15 cycles followed by terminalextension at 72° C. for 7 min.

The quantification of MAGE gene expression was conducted using aLightCycler instrument and the LightCycler FastStart DNA Master SYBRGreen I Kit (Roche Molecular Biochemicals, Mannheim, Germany). Theinherent fluorescence of the SYBR Green I dye is enhanced 200-fold whenit binds to the minor groove of double-stranded DNA. The increase influorescence is measured at the end of each cycle and indicates theamount of PCR products generated so far (F1, fluorescence channel 1 forSYBR Green I). Because of the labeling of any double-stranded DNAnonspecific PCR products. e.g. primer dimers, will contribute to thesignal. Therefore, the resulting PCR products in the SYBR Green Iprotocol are verified by means of a melting-curve analysis: since thedye only binds to double-stranded DNA, the fluorescent signal decreasesas the melting point of the DNA duplex is reached. Followingamplification the reaction mixture is subjected to an online meltingcurve analysis by increasing the temperature gradually (0.1° C./s).

The real-time PCR was carried out in 15 μl reaction mixture consistingof PCR grade water with 0.9 μl MgCl₂ (25 mM), 1 μl of each inner MAGEprimer (10 pmol/μl), 1.5 μl of FastStart DNA Master SYBR Green I and 4μl product of the first PCR reaction. Initial denaturation at 95° C. for5 min was followed by 45 cycles of denaturation at 95° C. for 15 s,annealing at 60° C. for 10 s and extension at 72° C. for 20 s with atemperature slope of 20° C./s performed in LightCycler capillaries. Allreactions were performed in duplicates and each run included a negativecontrol without a template.

The results of the amplification of the MAGE-A1 gene product are shownin FIG. 1: the amplification profile is depicted in FIG. 1A withfluorescence on the Y axis and the number of the PCR cycles on the Xaxis. The cycle number at which the amplification curve crosses thebaseline of the background signals is defined as crossing point and isused for quantification of the amount of template DNA in the sample. Asexpected more PCR cycles are required to amplify target cDNA in a samplecontaining less template cDNA. In this example it takes 22 cycles beforefluorescence can be detected in the blood sample spiked with 100 tumorcells, the crossing points for the samples contaminated with 10, 5 and 1cells follow consecutively. After 39 cycles there is also increase influorescence for the normal blood specimen, presumably caused byunspecific amplificates or primer-dimers.

FIG. 1B shows the negative derivatives of the melting curvecharacteristics at the end of the PCR reaction. The peaks represent themelting points of the PCR product, i.e. the temperature at which 50% ofthe DNA PCR product melted. The melting curves for the MAGE-A1 PCRproduct display a characteristic melting point at 88.8° C. Theunspecific amplificates in healthy blood, in this example primer dimers,show a different curve with a maximum at 87.5° C., enabling thediscrimination of different PCR products and verification ofspecificity. The gel electrophoresis in FIG. 1C reconfirmes the specificamplification of the MAGE-A1 product for 1 to 100 tumor cell in 2 ml ofblood.

The amplification of MAGE-A2 and -A12 did only yield specific signalswhen 10 tumor cells were added to 2 ml of blood. The increase offluorescence with lower cell numbers was due to unspecific products (seeFIGS. 2A and 2B).

Because of the lack of MAGE-A4 expression in Mz2-Mel cells we alsospiked 2 ml whole blood of healthy donors with different numbers ofLB-SAR cells that express MAGE-4 in a stable manner. The protocol wasthe same as described above but we used 2 μl of cDNA for PCR. Theresults of the amplification of the MAGE-4 gene product are shown inFIG. 3. Any samples with less than 5 tumor cells per 2 ml did not resultin specific detection of MAGE expression (FIG. 3A). For 5 and 10 cellsper 2 ml the real-time PCR yields the spefific MAGE-4 PCR product with amelting point of approximately 87.6° C., the melting curves for 0 and 1cell reveal the amplification of unspecific products (FIG. 3B).

To improve the sensitivity of the test we added 5 cycles to the protocolof the first PCR. The detection threshold was decreased thereby to 1tumor cell/2 ml of blood yielding specific MAGE-A4 products for alltumor cell dilutions (FIG. 4A). However, the increase in PCR cycles wasaccompanied by primer-dimer formation causing a melting peak for thenegative control without a template (FIG. 4B). A further addition of PCRcylces for preamplification was impossible because of the reaching of aplateau phase with impracticability of consecutive quantification ofgene expression.

This example shows the feasibility to detect the expression of singleMAGE genes by real-time PCR, but it moreover clearly demonstrates thedifficulty to ensure the amplification of all relevant MAGE mRNAspecies. Oligo-dT primed cDNA synthesis appears to prefer single mRNAmolecules while omitting others. This property leads to the completedrop out of several parameters with a dramatic decrease in sensitivityto detect rare transcripts. The addition of more cycles in the first PCRround cannot completely compensate this loss because of the increasingappearance of unspecific products. PCR methods using consensus MAGEprimers after reverse transcription with oligo-dT are especiallysusceptible for this considerable problem, because the putative“pan-MAGE” approach would only amplify some of the available MAGEtranscripts. Altough the standard protocol for reverse transcriptionusing oligo-dT primer is able to generate successful amplification ofMAGE-A1 transcipts it is not applicable for cDNA synthesis whenscreening for expression of the whole gene family.

EXAMPLE 2 Superiority of Highly Selected MAGE-Specific Primers forcDNA-Synthesis Prior to Amplification of MAGE by a Real-Time MultimarkerPCR

With the intention to establish a method for the sensitive detection ofall MAGE mRNA subtypes and simultaneously decrease unspecific backgroundwe modified the AMV reverse transcription protocol by using acombination of the outer antisense PCR primers for MAGE-1, -2, -3/6, -4and -12 in 2.5 μM solution for specific cDNA synthesis. Afterpreamplification with 20 cycles in a first PCR round the real-time PCRrevealed a deformation of the amplification plot (FIG. 5). Thepurification of the first PCR product led to the reconstitution of theregular amplification curve, presuming an overdosing of differentoligonucleotides in the cDNA synthesis that causes interference offluorescence in real-time PCR, although this approach could be shown towork in conventional PCR (Kufer 2002). The combination of severalantisense primers for RT recation seems not to be an applicable approachfor our purposes.

This accentuates the need for a specific shared oligonucleotide forreverse transcription of all MAGE-A mRNAs. We screened the MAGE-Asequences for universal segments by computer based sequence analysis andevaluated nine different oligonucleotides for their ability to act assensitive pan-MAGE primer for the detection of MAGE-A transcripts in a 2ml blood sample contaminated with 5 tumor cells (Mz2-Mel cells forMAGE-1, -2, -3/6 and -12; LB-SAR cells for MAGE-4) (Table 2). We usedthe standard 1^(st) strand cDNA Synthesis Kit for RT-PCR (AMV) suppliedby Roche Molecular Biochemicals (Mannheim, Germany) in 20 μl with 2 μlof 10× Reaction buffer, 5 mM MgCl₂, 1 mM of dNTP mixture, 50 units ofRNAse inhibitor, 20 units of AMV reverse transcriptase and 1 μg of RNAaccording to the manufacturer's protocol and added specificoligonucleotide primers in 2.5 μM concentration. The synthesis wasperfomed in a GeneAmp PCR System 9700 (Applied Biosystems, Foster City,USA) for 10 min at 25° C., 60 min at 42° C. and 5 min at 99° C. Only oneoligonucleotide turned out to be suitable for a reliable binding to allrelevant MAGE-A mRNAs (Table 3), the other candidates as well asOligo-dT, random hexamers or the combination of antisense PCR primerscaused the drop out of at least one marker or displayed low sensitivity(C_(T)-values>30). However, the transcription of the entire MAGE-A mRNAsby this single primer was accompanied by a late crossing-point forMAGE-A1 leading to poor sensitivity for this relevant marker.

In an attempt to improve the sensitivity for detection of the importantMAGE-A1 marker we evaluated several additional reverse transcriptionprimers specific for the synthesis of MAGE-A1 cDNA to be combined withthe prior established pan-MAGE primer within the same protocol (Table4). Again only one out of eleven combinations was able to amplify allrelevant MAGE mRNA species, the other compositions showed drop outs ofat least one marker (Table 5). Primer combinations failing to transcribeMAGE-A1 mRNA were excluded from further examination (“not determined” inthe table). Meanwhile we included the MAGE-A10 marker to the protocol asdecribed in example 4, nevertheless the combination of RT primers MgRT3aand Mg1_RT5a allowed the specific reverse transcription of all relevantMAGE-A mRNAs and the subsequent amplification in real-time PCR with muchhigher sensitivity than oligo-dT or random hexamers. This approachaccomplishes the cDNA synthesis of the complete MAGE transcriptome andis therefore the prerequisite for sensitive PCR assays, irrespective ofthe used primers or PCR strategy.

Here we could document the detection of all relevant MAGE markersexpressed in 5 tumor cells spiked into 2 ml of whole blood. Furthermorethe MAGE expression can be quantified using external standard curves ora direct comparison of single markers.

EXAMPLE 3 Quantification of MAGE Expression by Use of an InternalCalibrator

The reliable quantification of gene expression implies the inclusion ofan internal calibrator, e.g. a non-regulated housekeeping gene, toexclude variations in sample size or quality. Therefore we meant toquantify MAGE expression in relative comparison to the expression of ahousekeeping gene, e.g. porphobilinogen-desaminase (PBGD). For thispurpose we evaluated a broad variety of PBGD RT primers (Table 6) tointegrate the reverse transcription of PBGD mRNA into the establishedcDNA synthesis protocol described in example 2. Primers that failed toyield specific amplification of PBGD transcripts were excluded fromfurther evaluation. We tested 21 different PBGD specificoligonucleotides in combination with the prior established RT primersMgRT3a+Mg1_RT5a, but only one combination using the primer PBGD_RT10bresulted in the successful generation of specific PCR products of allexamined MAGE markers and PBGD (Table 7). However, the combination ofthree oligonucleotides in cDNA synthesis was linked to a markedlydecreased sensitivity (crossing-points >30 cycles for MAGE-2 and -12),thus appearing not to be an applicable approach when using real-time PCRsubsequently. Therefore we were forced to evaluate further protocolswith fewer than three RT primers.

Searching for more sensitive reverse transcriptase enzymes we tested thesingle pan-MAGE primer MgRT3a in two additional Kits for cDNA synthesisin a new experiment with 1 μg of total RNA from 2 ml of blood spikedwith 5 tumor cells (Mz2-Mel cells for MAGE-1, -2, -3/6, -10, -12 andLB-SAR cells for MAGE-4). The ThermoScript RT-PCR Kit supplied byInvitrogen (Groningen, Netherlands) completely failed to create anyamplifiable MAGE cDNA when we followed the standard protocol of themanufacture (Table 8), even though the Kit was designed for reversetranscription of difficult templates.

Additionally we applied an Omniscript RT Kit (Qiagen, Hilden, Germany)using the single pan-MAGE primer MgRT3a in 2.5 μM solution [with 2 μl of10× Buffer RT, dNTPs 0.5 mM each, 10 units of RNAse Inhibitor (Roche)and 4 units of Omniscript RT (Qiagen) for 60 min at 37° C. and 5 min. at93° C.] and could generate amplifyable cDNA of all relevant MAGE mRNAsand PBGD mRNA. The direct comparison of the Omniscript RT Kit to theprior used AMV Kit demonstrated a significantly higher sensitivity forthe Omniscript RT reaction without drop outs of single parameters whenanalyzing identical aliquots of the same RNA preparation (Table 8).

This new protocol was tested on it's compatibility with various PBGD RTprimers (Table 6) after the isolation of total RNA from 2 ml of bloodspiked with 10 tumor cells. Combinations that failed to generate apositive PBGD signal were excluded from further evaluation (quoted as“not determined” in the table). Only one out of 32 oligonucleotides(PBGD_RT15b) did not disturb the reverse transcription of MAGE mRNAswhile efficiently mediates the conversion of PBGD mRNA into cDNA (Table9). The second best candidate (PBGD_RT13a) already failed to generatethe same results when the sensitivity threshold was increased to 5 tumorcells/2 ml blood. The crossing-point (C_(T)) differences clearlyrepresent the varying levels of MAGE expression in the tumor cells withlow amounts of MAGE-A10 and -A12 transcripts and high quantity ofMAGE-A1 and -A4 mRNA in the utilized cell cultures as published in theliterature (Serrano, Lethe et al. 1999). The expression level of eachparticular MAGE gene can be quoted as C_(T) ^(MAGE)/C_(T) ^(PBGD) andcan therefore balance variations between individual samples. For thefirst time this protocol allows the reflection of the actual proportionsof MAGE gene expression in striking contrast to the prior art.

EXAMPLE 4 Detection of MAGE-A10 Transcripts Expressed by Rare TumorCells in Blood Using Real-Time MAGE RT-PCR

In the first evaluation of the MAGE genes it was assumed that MAGE-A10expression is only present at very low levels and therefore neglectableas marker for cancerous conditions. After the detection of MAGE-A10specific cytolytic lymphocytes (Huang LQ, Brasseur F et al. 1999) there-evaluation of expression profiles in tumor cells demonstrated weakbut frequent transcription of the MAGE-A10 gene (Serrano, Lethe et al.1999). Therefore it was our objective to include MAGE-A10 as anotheradditional sensitive marker in the described Multimarker MAGE RT PCR.

A selection of different sense and antisense primers specific forMAGE-A10 cDNA were designed (Table 10) and tested for their potential togenerate a MAGE-A10 PCR product. We isolated total RNA from 2 ml ofwhole blood contaminated with 100 Mz2-Mel cells and performed cDNAsynthesis with the MAGE-RT primers MgRT3a and Mg1RT5a as described inexample 2. The PCR was carried out in 50 μl reactions with 10×PCR buffer(Invitrogen), dNTP mixture 0.2 μM each (Invitrogen), 1.5 μM MgCl₂, 1.25units Platinum Taq DNA Polymerase (Invitrogen) and 2 μl of cDNA for 40cycles (initial enzyme activation at 95° C. for 3 min, denaturation at95° C. for 30 sec. annealing at 60° C. for 45 sec, elongation at 72° C.for 60 sec and final extension at 72° C. for 7 min). The PCR productswere analyzed by electrophoresis in a 30% polyacrylamide gel and stainedwith ethidium bromide. Several primer combinations yielded specificMAGE-A10 PCR products (Table 11A). The most promising combinations werefurther evaluated for application in real-time PCR using a LightCyclerFastStart SYBR Green I Kit. The reaction was performed in capillarieswith a total volume of 15 μl, 0.66 μM PCR primer each and 2.5 mM MgCl₂concentration for 50 cycles (initial enzyme activation at 95° C. for 5min, denaturation at 95° C. for 15 sec, annealing at 60° C. for 10 secand elongation at 72° C. for 20 sec). The results demonstrated 6successful primer combinations for the amplification of MAGE-A10 mRNA inthe LightCycler system (Table 11B) that were used to construct asensitive nested PCR for the detection of 5 Mz2-Mel cells in 2 ml ofblood. Four different combinations were tested in a first PCR round with20 cycles and subsequent real-time PCR analysis with the nested primerset Mg10_se3+Mg10_anse2. All experiments containing Mg10_anse4 asantisense primer in the first PCR round failed to yield specificamplification (FIG. 6), therefore primer sets including Mg10_se5 asantisense primer in the first PCR are the only possible composition. Thesense primers Mg10_se1 or Mg10_se3 can be successfully used for thefirst and second round of a nested or semi-nested PCR.

EXAMPLE 5 Analysis of Blood and Bone Marrow Samples of Cancer Patientswith a Highly Sensitive Multimarker MAGE Real-Time RT-PCR

After careful optimization of the reaction conditions we tested bloodand bone marrow samples from patients with localized prostate cancer fordisseminated tumor cells with the multimarker MAGE real-time RT-PCR. 1ml of bone marrow aspirate and 2 ml of blood were stabilized andprepared according to example 1. Total RNA was resuspended in 50 μl ofDEPC-treated water and 10 μl were utilized in the subsequent cDNAsynthesis using the Omniscript RT Kit (Qiagen, Hilden, Germany) in 20 μlwith 2 μl of 10× Buffer RT, 2 μl of the supplied dNTP mix, 1 μl ofMgRT3a (50 pmol/μl) and PBGD_RT15b primer (50 pmol/μl), 10 units ofRNAse inhibitor (Roche) and 4 units of Omniscript RT enzyme. Thereaction was executed in a GeneAmp PCR System 9700 (Applied Biosystems,Foster City, USA) for 60 min at 37° C. followed by denaturation at 93°C. for 5 min.

2 μl of the cDNA were used for the first round of PCR in 20 μl reactionswith 2 μl of 10×PCR Buffer, 0.6 μl MgCl₂ ₍50 μM), 1.6 μl dNTP mix and0.2 μl Platinum Taq DNA polymerase (all by Invitrogen, Groningen,Netherlands) and 0.4 μl of each outer MAGE primer (10 pmol/μl) accordingto the protocol described in example 1. For the real-time PCR weprepared 15 μl reactions in LightCycler capillaries with 1.5 μl ofFastStart DNA Master SYBR Green I reagent, 2 μl of the first PCR productand different concentrations of MgCl₂ and each inner MAGE primer: forMAGE-A1 we used 2.5 mM MgCl₂ and 1 μM inner MAGE-A1 primers, for MAGE-A2and -A10 3 mM MgCl₂ and 1.2 μM inner MAGE-A2 or -A10 primers, forMAGE-A3/6 and -4 2.5 mM MgCl₂ and 1.2 μM inner MAGE-A3/6 or -4 primers,and for MAGE-A12 3 mM MgCl₂ and 0.8 μM inner MAGE-A12 primers. Thereaction was run for 5 min at 95° C. for initial activation of theenzyme, 10 sec at 95° C. for denaturation, 5 sec at 60° C. for annealingand 10 sec at 72° C. for elongation for 40 cycles. After completion ofthe reaction the PCR products were subjected to a melting curve analysisspanning 65° C. to 95° C. with a ramping rate of 0.1° C./s and confirmedwith electrophoresis on 30% acrylamide gels in ambiguous cases.

The amplification of PBGD mRNA was performed in a separate real-time PCRin 20 μl with 1 μl of cDNA, 5 mM MgCl₂, 0.5 μM of sense primer (5′-AGAGTG ATT CGC GTG GGT ACC-3′), 0.5 μM of antisense primer (5′-TTG GGT GAAAGA CM CAG CAT C-3′) and 2 μl of FastStart DNA Master SYBR Green I(Roche). The protocol was modified as follows: initial enzyme activationfor 5 min at 95° C., denaturation for 15 sec at 95° C., annealing at 60°C. for 10 sec and extension for 20 sec at 72° C. After completion of thePCR the products were subjected to a melting curve analysis as describedbefore.

In total we were able to screen two groups of patients with prostatecancer after radical prostatectomy:

-   -   (a) 21 patients with attested biochemical relapse after radical        prostatectomy defined as rising serum PSA level >0.5 ng/ml in        the absence of any signs for local tumor growth. These patients        bear a high risk for developing metastatic disease because of        systemic spread of disseminated PSA producing tumor cells.    -   (b) 18 patients without biochemical relapse after radical        prostatectomy defined as serum PSA level <0.5 ng/ml and        presentation of a low risk profile for systemic tumor spread        (i.e. Gleason score 6 and preoperative serum PSA level 20 ng/ml        and tumor stage pT₁ or pT₂, pN₀, R₀) and postoperative        survival >30 months at the time of sample collection. In these        patients the development of metastatic disease should be an        unlikely event.

In the high risk group we could identify 14 patients (=66%, Table 12)with the expression of at least one MAGE gene in at least one sample(bilateral bone marrow aspirates or blood). The low risk group displayedMAGE expression in samples of 7 patients (=38%, Table 13). Furthermorethe total number of positive tests as well as the expression level wasmuch higher in the high risk than in the low risk cohort. It must beemphasized that the low risk group does not represent a true “negative”or “control” group, since these patients also had malignant disease.Because of the generally long disease free survival time of patientswith prostate cancer the detection of MAGE gene expression in the lowrisk group can be interpreted as true positive results with evidence forsystemic tumor spread before clinical manifestation of metastaticdisease at the present time. The quantity of MAGE expression can addfurther prognostic value, but the potency of these parameter has to beassessed after completion of the follow-up period of the cohort. Theanalysis of this cohort provides an impressive prove of principle forthe sensitive detection and quantification of several individual MAGEmarkers by the real-time RT-PCR of the present invention and theexemplified use of this method for early diagnosis of minimal residualtumor disease. TABLE 1 Members of the MAGE gene family showingrestricted expression in malignant tumors and testicular germ cellsonly. Gene Subfamily Gene Name MAGE-A hMAGE-A1 hMAGE-A2 hMAGE-A3hMAGE-A4 hMAGE-A5 hMAGE-A6 hMAGE-A8 hMAGE-A9 hMAGE-A10 hMAGE-A11hMAGE-A12 MAGE-B hMAGE-B1 hMAGE-B2 hMAGE-B3 hMAGE-B4 hMAGE-B5 hMAGE-B6hMAGE-B10 hMAGE-B16 hMAGE-B17 MAGE-C hMAGE-C1 hMAGE-C2 hMAGE-C3 hMAGE-C4

TABLE 2 Oligonucleotides used as primers for specific pan-MAGE reversetranscription primer sequence (5′ - 3′) MgRT1a CCA GCA TTT CTG CCT TTGTGA MgRT1b CCA GCA TTT CTG CCT GTT TG MgRT2 CAG CTC CTC CCA GAT TTMgRT3a ACC TGC CGG TAC TCC AGG MgRT3b ACC TGC CGG TAC TCC AGG TA MgRT4GCC CTT GGA CCC CAC AGG AA MgRT5a AGG ACT TTC ACA TAG CTG GTT TCA MgRT5bGGA CTT TCA CAT AGC TGG TTT C MgRT6 TTT ATT CAG ATT TAA TTT C

TABLE 3 Evaluation of different primers33for cDNA synthesis:Crossing-Points for the detection of MAGE mRNA by a multimarker MAGEreal-time RT-PCR. RT-Primer MAGE-1 MAGE-2 MAGE-3/6 MAGE-4 MAGE-12 random33.3 n.d. n.d.. n.d. n.d. hexamer Oligo-dT 30.1 — 27.0 30.2 — 3′ primer30.2 24.8 26.5 23.7 — combi- nation MgRT1a 28.8 — — 31.4 — MgRT1b — 22.020.9 35.1 — MgRT2 — 24.17 23.0 29.9 34.5 MgRT3a 30.2 22.7 19.8 21.7 22.1MgRT3b 28.7 — — 21.8 — MgRT4 — 23.4 22.4 20.7 — MgRT5a 27.6 — 20.6 22.4— MgRT5b 28.4 — — 20.0 — MgRT6 — — 21.6 — 25.0“n.d.” = not determined,“—” = negative signal

TABLE 4 Oligonucleotides used as34primers for MAGE-A1 specific reversetranscription primer sequence (5′ - 3′) Mg1_RT1 CAA GAG ACA TGA TGA CTCTC Mg1_RT2 TTC CTC AGG CTT GCA GTG CA Mg1_RT3 GAG AGG AGG AGG AGG TGG CMg1_RT4 GAT CTG TTG ACC CAG CAG TG Mg1_RT5a CAC TGG GTT GCC TCT GTCMg1_RT5c CTG GGT TGC CTC TGT CGA G Mg1_RT5d GGG TTG CCT CTG TCG AGT GMg1_RT5e GGC TGC TGG AAC CCT CAC Mg1_RT6 GCT TGG CCC CTC CTC TTC ACMg1_RT7 GAA CAA GGA CTC CAG GAT AC

TABLE 5 Combination of pan-MAGE RT35primer MgRT3a with different MAGE-A1specific RT primers for cDNA synthesis: Crossing-Points for thedetection of MAGE mRNA by a multimarker MAGE real-time RT-PCR.RT-Primer: MgRT3a+ MAGE-1 MAGE-2 MAGE-3/6 MAGE-4 MAGE-10 MAGE-12 Mg1outer 3′ 20.6 23.9 23.2 n.d. — — Mg1 RT1 19.8 — 21.7 n.d. n.d. — Mg1 RT2— 21.5 19.5 n.d. n.d. — Mg1 RT3 — n.d. n.d. n.d. n.d. n.d. Mg1 RT4 —n.d. n.d. n.d. n.d. n.d. Mg1 RT5a 20.7 22.1 12.1 20.9 23.5 23.5 Mg1 RT5c20.3 28.7 29.3 n.d. — — Mg1 RT5d 22.1 21.3 29.3 n.d. — — Mg1 RT5e 22.225.0 33.7 n.d. 24.1 22.8 Mg1 RT6 — n.d. n.d. n.d. n.d. n.d. Mg1 RT7 22.026.2 20.9 n.d. 25.9 —“n.d.” = not determined,“—” = negative signal

TABLE 6 Oligonucleotides used as36primers for specific reversetranscription of PBGD mRNA primer sequence (5′ - 3′) PBGD_-R TTG GGT GAAAGA CAA CAG CAT C PBGD_3 TTG CAG ATG GCT CCG ATG GTG AAG PBGD_3.1 R GGCTCC GAT GGT GAA GCC PBGD_RT1 AAC TCC TGC TGC TCG TCC AG PBGD_RT2 CAT ACATGC ATT CCT CAG GGT PBGD_RT3 GAA CTT TCT CTG CAG CTG GGC PBGD_RT4 TGGCAG GGT TTC TAG GGT CT PBGD_RT5 TTG TGC CAG CCC ATG CGC TG PBGD_10a GGTTTC CCC GAA TAC TCC TG PBGD_10b AGC TTC CGA AGC CGG GTG PBGD_10d TTG CTAGGA TGA TGG CAC TG PBGD_12a CTT GGC TCG CAC TTC CAC G PBGD_12b CCA AGATGT CCT GGT CCT TG PBGD_12c CAG CAC ACC CAC CAG ATC PBGD_12d AGA GTC TCGGGA TCG TGC PBGD_12e AGT CTC GGG ATC GTG CAG PBGD_12f TCT CGG GAT CGTGCA GCA PBGD_12g ATG CAG CGA AGC AGA GTC T PBGD_12h CCT TTC AGC GAT GCAGCG PBGD_13a GTA TGC ACG GCT ACT GGC PBGD_14a GCT ATC TGA GCC GTC TAG ACPBGD_14b GCA GGG ACA TGG ATG GTA G PBGD_15a AAT GTT ACG AGC AGT GAT GCPBGD_15b TGG GGC CCT CGT GGA ATG PBGD_15c AGC CAA CTG GGG CCC TCGPBGD_15d TAA GCT GCC GTG CAA CAT CC PBGD_15e CAG TTA ATG GGC ATC GTT AAGPBGD_15f ATC TGT GCC CCA CAA ACC AG PBGD_15g GGC CCG GGA TGT AGG CACPBGD_15h GGT AAT CAC TCC CCA GAT AG PBGD_15i CTC CCG GGG TAA TCA CTCPBGD_15j CAG TCT CCC GGG GTA ATC PBGD_15k TGA GGA GGC AAG GCA GTCPBGD_15l GGA TTG GTT ACA TTC AAA GGC

TABLE 7 Combination of primers MgRT3a and Mg1_RT5a with PBGD specific RTprimers for cDNA synthesis: Crossing-Points for the detection of PBGDand MAGE mRNA by a multimarker MAGE real-time RT-PCR. RT-Primer:MgRT3a + Mg1_RT5a+ PBGD MAGE-1 MAGE-2 MAGE-3/6 MAGE-4 MAGE-10 MAGE-12PBGD_R — — 24.5 24.9 20.9 n.d. 21.9 PBGD_3 — 20.3 21.8 20.1 n.d. n.d. —PBGD_3.1_R — — 22.6 20.2 n.d. n.d. — PBGD_RT1 — n.d. n.d. n.d. n.d. n.d.n.d. PBGD_RT2 14.3 22.3 21.8 — n.d. n.d. 28.7 PBGD_RT3 13.4 23.0 22.620.3 n.d. n.d. — PBGD_RT4 — n.d. n.d. n.d. n.d. n.d. n.d. PBGD_RT5 —n.d. n.d. n.d. n.d. n.d. n.d. PBGD_RT10a — n.d. n.d. n.d. n.d. n.d. n.d.PBGD_RT10b 13.0 23.2 31.6 21.3 n.d. 24.6 33.3 PBGD_RT10d 14.1 22.7 23.821.1 n.d. — 30.0 PBGD_RT12a — n.d. n.d. n.d. n.d. n.d. n.d. PBGD_RT12b14.4 29.0 21.6 32.0 n.d. — — PBGD_RT12c 14.6 28.0 20.8 >36   n.d.— >36   PBGD_RT12d 15.2 26.0 22.3 >36   n.d. — >36   PBGD_RT12e 14.326.8 23.2 21.2 n.d. — >36   PBGD_RT12f 14.6 21.2 — 21.9 n.d. — —PBGD_RT12g 14.9 21.8 29.4 23.3 n.d. — 22.6 PBGD_RT12h 15.2 — 22.7 21.5n.d. — — PBGD_RT13a 14.0 — 23.5 — n.d. — 24.5 PBGD_RT15a 14.5 21.9 25.820.9 n.d. — 31.3“n.d.” = not determined,“—” = negative signal

TABLE 8 Evaluation of different reverse transcriptase enzymes:Crossing-Points for the detection of PBGD and MAGE mRNA by a multimarkerMAGE real-time RT-PCR. RT Enzyme: MAGE-1 MAGE-2 MAGE-3/6 MAGE-4 MAGE-10MAGE-12 Thermoscript — — — n.d. — — (Invitrogen) AMV — 26.0 19.4 n.d. —22.3 (Roche) Omniscript 19.6 22.1 19.8 16.9 23.8 21.5 (Qiagen)“n.d.” = not determined,“—” = negative signal

TABLE 9 Combination of pan-MAGE RT 42primer MgRT3a with different PBGDRT primers using the Omniscript RT protocol: Crossing-Points for thedetection of PBGD and MAGE mRNA by a multimarker MAGE real-time RT-PCR.RT Primer: MgRT3a+ PBGD 43 MAGE-1 MAGE-2 MAGE-3/6 MAGE-4 MAGE-10 MAGE-12PBGD_R — n.d. n.d. n.d. n.d. n.d. n.d. PBGD_RT1 — n.d. n.d. n.d. n.d.n.d. n.d. PBGD_RT2 22.7 — n.d. n.d. n.d. n.d. — PBGD_RT3 18.9 — n.d.n.d. n.d. n.d. — PBGD_RT4 19.8 — n.d. n.d. n.d. n.d. — PBGD_RT5 — n.d.n.d. n.d. n.d. n.d. n.d. PBGD_RT10a 16.3 — n.d. n.d. n.d. n.d. —PBGD_RT10b — n.d. n.d. n.d. n.d. n.d. n.d. PBGD_RT10d 22.6 — n.d. n.d.n.d. n.d. — PBGD_RT12a — n.d. n.d. n.d. n.d. n.d. n.d. PBGD_RT12b 18.0 —n.d. n.d. n.d. n.d. — PBGD_RT12c 21.6 21.0 — 32.2 n.d. 35.7 — PBGD_RT12d25.6 21.6 25.1 30.1 n.d. — — PBGD_RT12e 18.6 22.2 23.0 25.8 n.d. — —PBGD_RT12f 20.8 22.8 — 30.1 n.d. — — PBGD_RT12g 22.9 — n.d. n.d. n.d.n.d. — PBGD_RT12h 23.6 20.3 22.1 26.7 n.d. — — PBGD_RT13a 21.4 21.1 23.720.1 n.d. 30.0 25.6 PBGD_RT14a 23.7 22.8 29.2 27.0 n.d. — >33  PBGD_RT14b — — n.d. n.d. n.d. n.d. — PBGD_RT15a 23.8 19.7 25.9 22.0 n.d.— 24.6 PBGD_RT15b 20.2 20.3 22.2 28.9 23.1 25.5 21.7 PBGD_RT15c — n.d.n.d. n.d. n.d. n.d. n.d. PBGD_RT15d — — n.d. n.d. n.d. n.d. — PBGD_RT15e18.5 20.3 22.0 33.0 n.d. — 23.9 PBGD_RT15f 19.9 19.8 27.0 18.0 n.d. 22.6— PBGD_RT15g 18.5 21.2 23.3 29.0 n.d. — — PBGD_RT15h 22.7 — n.d. n.d.n.d. n.d. — PBGD_RT15i 22.0 — 26.2 19.8 n.d. — — PBGD_RT15j 24.6 18.820.7 25.7 n.d. — — PBGD_RT15k 22.7 18.7 20.0 26.5 n.d. — 22.3 PBGD_RT15l24.2 — 21.8 26.6 n.d. 23.2 19.6“n.d.” = not determined,“—” = negative signal

TABLE 10 Oligonucleotides used as PCR primers for the amplification ofMAGE-A10 cDNA primer sequence (5′ - 3′) Mg10_se1 CTA CAG ACA CAG TGG GTCGC Mg10_se2 GCA GGA TCT GAC AAG AGT CC Mg10_se3 ATC TGA CAA GAG TCC AGGTCC Mg10_anse1 TGG GAG TGT GGG CAG GAC T Mg10_anse2 CGC TGA CGC TTT GGAGCT C Mg10_anse3 ATC CTC CTC CAC AGC CAG G Mg10_anse4 GGA GCT GGT GGAAGT GGA TG Mg10_anse5 GCT TGG TAT TAG AGG ATA GCA G Mg10_anse6 CAT CAGCAG AAA CCT CCT CTG Mg10_anse7 AAT GGA AGG GAA GCA ACG ACC Mg10_anse8GGA GCC CTC ATC AGA TTG ATC

TABLE 11 Combination of MAGE-A1046specific sense and antisense PCRprimer primer combination Mg10_se1+ Mg10_se2+ Mg10_se3+ Results ofconventional RT-PCR with 40 cycles using total RNA isolated from 2 ml ofblood spiked with 100 Mz2/Mel cells. Mg10_anse1 − − + Mg10_anse2 + − +++Mg10_anse3 − − + Mg10_anse4 ++ + ++ Mg10_anse5 ++ + ++ Mg10_anse6 + + +Mg10_anse7 + (+) + Mg10_anse8 − − + Results of real-time PCR with 50cycles using total RNA isolated from 2 ml of blood spiked with 100Mz2/Mel cells: Crossing-Points for the detection of MAGE-A10 mRNA by amultimarker MAGE real-time RT-PCR Mg10_anse2 n.d. 37.4 Mg10_anse4 33.735.8 Mg10_anse5 38.9 39.9 Mg10_anse6 n.d. 40.8“(+)” - “+++” = intensity of specific signal, “−” = no signal

TABLE 12 Crossing-Points for the47detection of PBGD and MAGE mRNA bymultimarker MAGE real-time RT-PCR in blood and bone marrow aspirates ofpatients with confirmed relapse of prostate cancer. patient CK-ICCsamples PBGD MAGE-1 MAGE-2 MAGE-3/6 MAGE-4 MAGE-10 MAGE-12 1. neg BMright 26.1 — — — — — — BM left 26.6 — — — — — — Blood 22.4 — — — — — —2. pos BM right 26.2 — — — — — — BM left 24.8 — — — — — — Blood 21.7 —24.5 — — — — 3. neg BM right 23.8 — — — — — — BM left 23.8 — — — — — —Blood — — — — — — — 4. neg BM right 21.8 — 24.5 — — — — BM left 23.6 — —— — — — Blood 22.6 — — — — — — 5. neg BM right 20.8 — 23.9 — — — — BMleft 24.2 — — — — — — Blood 23.3 — — >36   — — — 6. neg BM right 23.5 —— — — — — BM left 21.9 — — — — — — Blood 21.7 — — — — — — 7. neg BMright 26.2 — — — — — — BM left 24.2 — — — — — — Blood 20.0 — — — — — —8. neg BM right 28.2 23.7 11.8 — 33.0 — — BM left 26.9 — — — — — — Blood22.7 — — — 29.4 — — 9. pos BM right 24.1 — — — — — — BM left 25.0 — — —— — — Blood 23.1 — — — — — — 10. pos BM right 20.9 — — — — — — BM left20.2 — — — — — — Blood 24.9 — — — — — — 11. neg BM right 22.8 — — — 29.2— — BM left 21.2 — — — — — — Blood 22.4 — — — — — — 12. neg BM right27.3 — — — — — — BM left 24.7 — — — — — — Blood 21.6 — — — — — — 13. negBM right — — — — — — — BM left 27.0 19.1 — — — — — Blood 23.1 — — — — —— 14. neg BM right 24.3 13.2 — — — — — BM left 24.1 12.6 23.8 — — — —Blood 24.1 — — — — — — 15. neg BM right 24.1 <7.0 23.6 — >36   — — BMleft 24.0  7.2 — — — — — Blood 22.6 — — — — — — 16. neg BM right — — — —— — — BM left 25.5 18.5 28.8 — >36   — — Blood 22.1 — — — — — — 17. negBM right 23.0 — — >36   29.4 — — BM left 29.5 — — 19.5 — — — Blood 22.6— — — — — — 18. neg BM right 26.1 — — 19.3 — — 22.0 BM left 24.6 — — — —— — Blood 21.8 21.2 27.5 — — — — 19. neg BM right 24.2 — — — 16.7 — — BMleft 25.0 — — — — — — Blood n.a. 20. neg BM right 24.1 — — — — — — BMleft 26.7 — — — — — — Blood 21.5 — — — — 32.4 — 21. neg BM right 23.0 —— — 10.6 30.9  9.8 BM left 30.3 — — 21.0 <7.0 — 13.1 Blood 20.7 — — — —— —CK-ICC = cytokeratin-immunocytochemistry,BM = bone marrow aspirate

TABLE 13 Crossing-Points for the49detection of PBGD and MAGE mRNA bymultimarker MAGE real-time RT-PCR in blood and bone marrow aspirates ofpatients with low risk for relapse of prostate cancer. patient CK-ICCsamples PBGD MAGE-1 MAGE-2 MAGE-3/6 MAGE-4 MAGE-10 MAGE-12 1. pos BMright 20.0 — — — — — — BM left 21.6 — — — — — — Blood 23.0 — — — — — —2. neg BM right 20.4 — — — — — — BM left 19.4 — — — — — — Blood 20.1 — —— — — — 3. neg BM right 29.0 — — — — — — BM left 27.1 — — — — — — Blood29.6 — — — — — — 4. neg BM right 26.5 — — — — — — BM left 24.7 — — — — —— Blood 21.2 — — — — — — 5. neg BM right 25.2 21.4 — — — — — BM left25.7 21.8 — — — — — Blood 22.3 — — — — — — 6. neg BM right 26.8 — — — —— — BM left 27.3 — — — — — — Blood 23.7 — — — >36   — — 7. pos BM right22.0 — — — — — — BM left 21.7 — — — — — — Blood 22.9 — — — — — — 8. negBM right 23.1 — — — — — — BM left 22.2 — — — — — — Blood 22.2 — — — — —— 9. neg BM right 35.5 — — — 30.1 — — BM left — — — — — — — Blood 22.1 —— — — — — 10. neg BM right 24.1 — — — — — — BM left 29.6 — — — — — —Blood 22.8 — — — — — — 11. neg BM right 23.4 — — — — — — BM left 22.7 —— — — — — Blood 22.7 — — — — — — 12. neg BM right — — — — — — — BM left24.0 — — — — — — Blood 23.5 — — — >36   — — 13. neg BM right >36   — — —— — — BM left 25.0 18.9 — — 20.6 — — Blood n.a. 14. neg BM right 27.623.9 — — — — — BM left 24.8 — — — — — — Blood 24.4 — — — — — — 15. negBM right 30.4 — — — — — — BM left 26.9 — — — — — — Blood 21.5 — — — — —— 16. neg BM right — — — — — — — BM left 23.2 — — — — — — Blood 21.9 — —— — — — 17. neg BM right 26.3 14.7 18.5 — 28.2 — 29.6 BM left 31.2 13.2— — 20.8 — — Blood 21.8 — — — >36   — — 18. neg BM right 24.3 — — — — —— BM left 24.6 — — — — — — Blood 21.2 — — — — — —CK-ICC = cytokeratin-immunocytochemistry,BM = bone marrow aspirate

1. A highly sensitive real-time RT-PCR capable of specifically detectingthe expression of more than one MAGE gene, wherein reverse transcriptionof the corresponding MAGE transcripts is carried out simultaneously in asingle cDNA-synthesis reaction.
 2. The method of claim 1, wherein theMAGE genes are selected from the functional genes of MAGE subfamilies A,B and/or C.
 3. The method of claim 1, wherein the selected MAGE genescomprise MAGE-A 1, 2, 3, 4, 6, 10 and/or
 12. 4. The method of claim 1,wherein at least one primer for reverse transcription of MAGE mRNA isselected from the following groups of oligonucleotides: Primer Sequence(5′ - 3′) SEQ ID NO (A) MgRT1a CCA GCA TTT CTG CCT TTG TGA 1 MgRT1B CCAGCA TTT CTG CCT GTT TG 2 MgRT2 CAG CTC CTC CCA GAT TT 3 MgRT3a ACC TGCCGG TAC TCC AGG 4 MgRT3b ACC TGC CGG TAC TCC AGG TA 5 MgRT4 GCC CTT GGACCC CAC AGG AA 6 MgRT5a AGG ACT TTC ACA TAG CTG GTT TCA 7 MgRT5b GGA CTTTCA CAT AGC TGG TTT C 8 MgRT6 TTT ATT CAG ATT TAA TTT C 9 (B) Mg1_RT1CAA GAG ACA TGA TGA CTC TC 10 Mg1_RT2 TTC CTC AGG CTT GCA GTG CA 11Mg1_RT3 GAG AGG AGG AGG AGG TGG C 12 Mg1_RT4 GAT CTG TTG ACC CAG CAG TG13 Mg1_RT5a CAC TGG GTT GCC TCT GTC 14 Mg1_RT5c CTG GGT TGC CTC TGT CGAG 15 Mg1_RT5d GGG TTG CCT CTG TCG AGT G 16 Mg1_RT5e GGC TGC TGG AAC CCTCAC 17 Mg1_RT6 GCT TGG CCC CTC CTC TTC AC 18 Mg1_RT7 GAA CAA GGA CTC CAGGAT AC 19


5. The method of claim 1, wherein in addition to the reversetranscription of MAGE transcripts reverse transciption of a calibratormRNA is simultaneously carried out in the same single cDNA-synthesisreaction followed by PCR-amplification of MAGE- and calibrator cDNAs. 6.The method of claim 5, wherein the calibrator mRNA is porphobilinogendesaminase (PBGD), glyceraldehyd-3-phospat dehydrogenase (GAPDH),beta-2-microglobin or beta-actin.
 7. The method of claim 6, wherein theprimer for reverse transcription of PBGD mRNA is selected from thefollowing group of oligonucleotides: Primer Sequence (5′ - 3′) SEQ ID NOPBGD_RT2 CAT ACA TGC ATT CCT CAG GGT 20 PBGD_RT3 GAA CTT TCT CTG CAG CTGGGC 21 PBGD_RT4 TGG CAG GGT TTC TAG GGT CT 22 PBGD_RT10a GGT TTC CCC GAATAC TCC TG 23 PBGD_RT10d TTG CTA GGA TGA TGG CAC TG 24 PBGD_RT12b CCAAGA TGT CCT GGT CCT TG 25 PBGD_RT12c CAG CAC ACC CAC CAG ATC 26PBGD_RT12d AGA GTC TCG GGA TCG TGC 27 PBGD_RT12e AGT CTC GGG ATC GTG CAG28 PBGD_RT12f TCT CGG GAT CGT GCA GCA 29 PBGD_RT12g ATG CAG CGA AGC AGAGTC T 30 PBGD_RT12h CCT TTC AGC GAT GCA GCG 31 PBGD_RT13a GTA TGC ACGGCT ACT GGC 32 PBGD_RT14a GCT ATC TGA GCC GTC TAG AC 33 PBGD_RT15a AATGTT ACG AGC AGT GAT GC 34 PBGD_RT15b TGG GGC CCT GCT GGA ATG 35PBGD_RT15e CAG TTA ATG GGC ATC GTT AAG 36 PBGD_RT15f ATC TGT GCC CCA CAAACC AG 37 PBGD_RT15g GGC CCG GGA TGT AGG CAC 38 PBGD_RT15h GGT AAT CACTCC CCA GAT AG 39 PBGD_RT15i CTC CCG GGG TAA TCA CTC 40 PBGD_RT15j CAGTCT CCC GGG GTA ATC 41 PBGD_RT15k TGA GGA GGC AAG GCA GTC 42 PBGD_RT15lGGA TTG GTT ACA TTC AAA GGC 43


8. The method of claim 5, wherien the PCR-primers for amplification ofPBGD-cDNA comprises oligonucleotides selected from the following groups:SEQ ID PBGD Sequence (5′ - 3′) NO Sense Primer Hu_PBGD_se AGA GTG ATTCGC GTG GGT ACC 44 PBGD_8 GGC TGC AAC GGC GGA AGA AAA C 45 PBGD_8_F TGCAAC GGC GGA AGA AAA C 46 PBGD_ATG-Eco ATG TCT GGT AAC GGC AAT GC 47Antisense Primer PBGD_3 TTG CAG ATG GCT CCG ATG GTG A 48 PBGD_3.1_R GGCTCC GAT GGT GAA GCC 49 PBGD_R TTG GGT GAA AGA CAA CAG CAT C 50


9. The method of claim 8, wherein oligonucleotides hu PBGD se and PBGD3.1 R or hu PBGD se and PBGD R are used as primer pairs forPCR-amplification of PBGD-cDNA.
 10. The method of claim 1, wherein intotal not more than two different oligonucleotides are used as primersfor reverse transcription in the cDNA-synthesis reaction.
 11. The methodof claim 10, wherein the oligonucleotides MgRT3a and/or Mg1 RT5a areused as primers for reverse transcription in the cDNA-synthesisreaction.
 12. The method of claim 10, wherein the oligonucleotidesMgRT3a and PBGD RT15bare used as primers for reverse transcription inthe cDNA-synthesis reaction.
 13. The method of claim 1, wherein theMAGE- and/or the calibrator-PCR are nested or semi-nested PCRs.
 14. Themethod of claim 1, wherein PCR-primers are used comprising pairs ofoligonucleotides specifically amplifying only a single member of theselected group of MAGe genes, respectively.
 15. The method of claim 1,wherein PCR-primers are used comprising pairs of oligonucleotidescomprising pairs of PCR-primers amplifying more than one member of theselected group of MAGE genes, respectively.
 16. The method of claim 1,wherein the PCR-primers for amplification of MAGE-cDNA compriseoligonucleotides selected from one of the following groups: PCR-PrimerSequence (5′ - 3′) SEQ ID NO (C) MAGE-A1 GTA GAG TTC GGC CGA AGG AAC 51MAGE-A1 CAG GAG CTG GGC AAT GAA GAC 52 MAGE-A2 CAT TGA AGG AGA AGA TCTGCC T 53 MAGE-A2 GAG TAG AAG AAG AAG CGG T 54 MAGE-A3/6 GAA GCC GGC CCAGGC TCG 55 MAGE-A3/6 GAT GAC TCT GGT CAG GGC AA 56 MAGE-A4 CAC CAA GGAGAA GAT CTG CCT 57 MAGE-A4 TCC TCA GTA GTA GGA GCC TGT 58 MAGE-A10 CTACAG ACA CAG TGG GTC GC 59 MAGE-A10 GCT TGG TAT TAG AGG ATA GCA G 60MAGE-A12 TCC GTG AGG AGG CAA GGT TC 61 MAGE-A12 ATC GGA TTG ACT CCA GAGAGT A 62 (D) MAGE-A1 TAG AGT TCG GCC GAA GGA AC 63 MAGE-A1 CTG GGC AATGAA GAC CCA CA 64 MAGE-A2 CAT TGA AGG AGA AGA TCT GCC T 65 MAGE-A2 CAGGCT TGC AGT GCT GAC TC 66 MAGE-A3/6 GGC TCG GTG AGG AGG CAA G 67MAGE-A3/6 GAT GAC TCT GGT CAG GGC AA 68 MAGE-A4 CAC CAA GGA GAA GAT CTGCCT 69 MAGE-A4 CAG GCT TGC AGT GCT GAC TCT 70 MAGE-A10 ATC TGA CAA GAGTCC AGG TTC 71 MAGE-A10 CGC TGA CGC TTT GGA GCT C 72 MAGE-A12 TCC GTGAGG AGG CAA GGT TC 73 MAGE-A12 GAG CCT GCG CAC CCA CCA A 74


17. The method of claim 16, wherein primers of group C are used for afirst round and/or primers of group D for a second round ofPCR-amplification.
 18. The method of claim 15 carried out with a singleor double pair of PCR-primers amplifying all members of the selectedgroup of MAGE genes, respectively.
 19. A diagnostic compositioncomprising one or more suitable cDNA-primers for simultaneous reversetranscription of more than one different MAGE gene transcripts andoptionally an appropriate calibrator mRNA in a single cDNA-synthesis.20. The diagnostic composition of claim 19, wherein at least onecDNA-primer is MgRT3a, Mg1_RT5a or PBGD_RT15b.
 21. An oligonucleotideselected from the following group of primers: MgRT3a Mg1_RT5a PBGD_RT15b